Use of metal compounds to treat gastrointestinal infections

ABSTRACT

Cobalt salts have been found to be particularly effective against  H. pylori  and may therefore be used to treat gastrointenstinal infection with this bacteria. The cobalt salts have the advantage of showing a good degree of selectivity for  H. pylori  over other Gram positive and Gram negative bacteria. Treatment with the cobalt salts may be carried out at the same time as conventional treatment with an antibiotic and/or a proton pump inhibitor.

This invention relates to the use of cobalt salts to treat or preventgastrointestinal infections. The invention also relates topharmaceutical compositions comprising a cobalt salt and which areadapted for controlled release of cobalt ions or which comprise anantibiotic and/or a proton pump inhibitor and to methods of treating orpreventing gastrointestinal infections using cobalt salts.

Gastrointestinal infections cause widespread diarrhoea and debility andaccount for a large proportion of antibiotic use worldwide. Thenon-specificity of antibiotics has meant that resistant pathogens are anincreasing problem leading to more complex treatments. Furthermore, manyantibiotics have side effects that reduce compliance, while cost maypreclude their use in developing countries where infections are morecommon. Even in the western world, complex treatment is often required,for example, H. pylori infection of the gastric mucosa requires “tripletherapy” for successful eradication. There are however few gut-specificantimicrobials so antibiotics designed for absorption and systemicaction are mainly used.

As early as the end of the nineteenth century, spiral organisms wereobserved in animal stomachs. Although identical observations were madein human biopsy specimens in 1906, it was not until 1975 that thepresence of this bacterium was first associated with gastritis. Shortlyafter this, association of the bacterium with gastritis, peptic ulcersand some forms of gastric cancer were confirmed. The bacterium waseventually named Helicobacter pylori (H. pylori) and other Helicobacterspecies have since been isolated from animal stomachs giving rise to anentirely new genus. Half of the western population over 60 years old andmost of the developing world population is infected with H. pylori.Although only a minority of infected people are symptomatic thebacterium is now a well recognised risk factor for gastric cancer so itseradication at the population level is a major goal of modern medicine.

H. pylori is a Gram negative bacterium that lives within and under themucus layer on the surface of the mucosa in the human stomach where itcan trigger inflammation. Bacteria were once thought not to be able tocolonise this environment due to pH change and mucus shedding. However,H. pylori is specifically adapted to this constantly changingenvironment.

The sequence of events that leads to the establishment of H. pyloriinfection is still only partly understood. The helical shape and theflagellae provide H. pylori with high motility even in the viscousgastric environment. Also, segments of lipopolysaccharades of the outermembrane of H. pylori have the ability to mimic the Lewis^(x) and/orLewis^(y) blood group antigen, thus camouflaging the bacterium andevading host defences. However, the most important feature of H. pylorithat allows colonisation and survival in the stomach environment is thehigh rate of production of the urease enzyme. Urease catalyses thedegradation of urea into carbon dioxide and ammonia, and is present bothinternally and externally (from cell lysis). It was first believed thatthe ammonia released by external urease (creating a cloud in theimmediate surrounding of the bacteria) protected H. pylori from acidattack, but, it was later discovered that under acidic conditions theinternal urease maintains a tolerable periplasmic pH, allowing proteinsynthesis and bacterial growth (Scott D et al, The role of internalurease in acid resistance of Helicobacter pylori. Gastroenterology,114:58-70 1998).

H. pylori infection causes inflammation of the gastric mucosa (i.e.gastritis) by both directly affecting epithelial cells and by inducingan inflammatory response. The distribution and severity of gastritisvaries widely. Most people with H. pylori infection are asymptomatic,but symptoms vary from dyspepsia (upper abdominal discomfort with orwithout nausea, vomiting and burning sensations in the epigastrum) topeptic ulcers and gastric cancer. Duodenal ulcer and gastric cancer seemto be mutually exclusive outcomes of H. pylori infection. It is nowrecognized that antral-predominant H. pylori gastritis stimulatesincreased release of gastrin from the antral mucosa which results inexcessive secretion of acid by the parietal cells in the healthy,uninflamed body mucosa. This increased acid secretion results in anincreased duodenal acid load and gastric metaplasia of the duodenalbulb. H. pylori can colonise this gastric metaplasia, and so thecombination of both increased acid load and the colonising organisminduce duodenal mucosal damage and ulceration (McColl, Helicobacterpylori 1988-1998. European journal of gastroenterology and hepathology,11:13-16 1999). In contrast, gastritis involving the acid-secretingcorpus region of the stomach leads to hyposecretion of gastric acid,eventually leading to achlorhydria, which is associated with anincreased risk of gastric cancer (El-Omar et al, Interleukin-1polymorphisms associated with increased risk of gastric cancer, Nature,404:398-402 2000). The nature of this relationship is however stillunclear.

H. pylori strains expressing the vacA gene, responsible for thesecretion of the cytotoxin VacA that causes the formation of vacuoles inmammalian cells in vitro, are also associated with more severepathologies. In addition, the severity of gastric inflammationcorrelates with cagA gene expression which is responsible for thesecretion of the cytotoxin-associated protein CagA that can induce celldeath (El-Omar et al., 2000). However, these virulence factors cannotexplain the difference in clinical outcome described above, since bothduodenal ulcer and gastric cancer are associated with vacA and cagAexpression. Host genetic factors, and environmental factors such asdiet, may explain the different responses to H. pylori infection.Recently, genetic factors affecting the production of interleukin-1-β,an important pro-inflammatory cytokine and a powerful inhibitor of acidsecretion, have been implicated in the development of gastric cancer(El-Omar et al, 2000).

The finding that H. pylori infection is a marked risk factor forsubsequent development of gastric cancer means that infection is rarelynow treated conservatively. Eradication of H. pylori in infectedindividuals, whether symptomatic or not, is increasingly common.Antibiotics form the basis of all treatments although neither mono nordual therapies have ever been especially efficacious. The addition ofbismuth compounds or antisecretory drugs to antibiotic dual therapies,forming the commonly used triple therapies, has led to significantlyimproved eradication rates (typically 70-95%). Quadruple therapies havealso been used, consisting of two antibiotics in combination with anacid suppressing agent and a bismuth compound. However, due to theircomplexity and an undesirable interaction between bismuth and acidsuppressing agents, these therapies have been restricted to second linetreatment for resistant H. pylori infection. Recently, efforts haveconcentrated on the reduction of doses and duration of therapy toimprove compliance, side effects and costs. Nonetheless, we are stillleft with mass prescription of antibiotics for one of the world's mostprevalent infections. As a result antibiotic resistance is becomingincreasingly common.

Prior to the discovery of H. pylori the main treatment of gastric andpeptic ulcers was the administration of inhibitors of acid secretion,namely H2-antagonist and proton pump inhibitors (PPI). Following theestablishment of the role of H. pylori in such diseases, the addition ofantisecretory agents was found to enhance the eradication rates of allthe antibiotics.

During antisecretory therapy H. pylori tends to migrate from the antrumto the less alkaline corpus which may also favour its eradication.Nevertheless, acid inhibition increases the bioavailability ofantibiotics by increasing the pH of the gastric juice, which firstincreases their concentration and secondly stabilises the active form.In addition, PPI may increase direct delivery of antibiotics to themucosa by decreasing gastric mucus viscosity in vivo.

In vitro, H. pylori is naturally susceptible to most antibiotics but,unfortunately, no single antibiotic therapy achieves high eradicationrates in vivo. This could either be due to extrinsic factors (extent ofinfection, immune status of individuals and compliance with treatmentregimens), or intrinsic factors (biochemically and genetically basedmicrobial resistance) (Hoffman, Antibiotic resistance mechanisms ofHelicobacter pylori, Canadian journal of gastroenterology, 13(3):243-2491999).

Antibiotics, which are usually administered orally to eradicate H.pylori infection, are predominantly absorbed across the intestinallumen, and their subsequent gastric secretion is not clearly defined.The availability and stability of antibiotics at the site of infectiondepends upon their pKa, lipid solubility and complexation by protein, aswell as the mucosal pH gradient.

Among the many antibiotics examined in vitro, only four are clinicallyuseful: amoxycillin, tetracycline, the newer macrolides (of whichclarithromycin is best studied, although others are under investigation)and nitroimidazoles (usually metronidazole). Clarithromycin, which isless affected by a decrease in pH than that of other compounds, has avery low MIC for H. pylori (MIC50, 0.03 mg.L-1). As a monotherapy,clarithromycin eradicates only about 34% of infections which rises toabout 70% in combination with metronidazole. In triple therapy with aPPI and either metronidazole or amoxycillin this increases to 85-95%(Hoffman 1999). The frequency of resistance to clarithromycin (i.e.macrolides) varies from country to country and seems to parallel the useof this therapeutic class to treat other infections (mainly respiratorytract infection). Similarly, it is of concern that prescription ofclarithromycin for H. pylori could lead to resistance of otherpathogenic bacteria. A marked difference has also been found between therates of resistance to nitroimidazoles in developed (10-50%) anddeveloping (80-90%) countries, related to their extent of use in otherinfections such as parasitic, genital, and dental infections.

Resistance to these antibiotics significantly affects their efficacy inbacterial eradication. However, Clarithromycin resistance, for example,alters the efficacy of antisecretory based triple therapy withamoxicillin and clarithromycin, eradication changing from 94%(susceptible) to 45% (resistant). There has not been any report ofresistance to the β-lactam amoxycillin in H. pylori infection but H.pylori strains have been described that repeatedly showed higher MIC(0.25 or 0.5 mg. L-1 instead of usually ≦0.03 mg.L-1). One of the fourpenicillin-binding proteins that are normally present in susceptiblestrains was missing in isolates with higher MICs. Currently, thisphenomenon seems rare, but it nevertheless indicates the need forsurveillance.

Antibiotic resistance may also be linked to compliance because, when apatient takes a course of drugs incompletely, there is the possibilitythat the concentration of antibiotic at the site of infection decreasesto a level where resistance emerges; an increase in resistance is thenanticipated.

Toxic metal compounds have been in use for some considerable time in thetreatment of gastrointestinal symptoms and of gastrointestinal and evensystemic infections, but significant side effects occur, such as theencephalopathy seen with bismuth complexes (Gorbach S. L.,Gastrenterology, 99: 863-875 (1990)). Newer “Colloidal” bismuthcompounds such as De-Noltab™ (bismuth sub-citrate) and Pepto-Bismol™(bismuth sub-salicylate) are less well absorbed in man and have someactivity against gastrointestinal bacteria. However, it has still beenshown that significant and prolonged plasma levels of bismuth are foundfollowing ingestion of such colloidal preparations (Nwokolo et al,Alimentary, Pharmacology and Therapeutics, 4:163-169 (1990)) (reachingup to 135 μg/l for De-Noltab™ and 5 μg/l for Pepto Bismol™).

These earlier metal-based therapies in the gastrointestinal tract have,unknowingly, been mainly effective against gastrointestinal pathogensdue to their synergistic action with conventional antibiotics, ratherthan due to any significant antimicrobial properties per se, as unlikein vitro, sufficient concentrations of bismuth may not reach thebacteria in vivo.

This has been suggested by work we have carried out on the therapeuticrole of bismuth compounds in the eradication of H. pylori.

The microbial infections treatable by the methods described hereinrelate to H. pylori. H. pylori is a Gram negative bacteria that has beenstrongly implicated in chronic active gastritis and peptic ulcer disease(Marshall et al, Medical Journal of Australia, 142:439-444 (1985); Buck,G. E., Journal of Clinical Microbiology, 3:1-12 (1990)). More recently,it has also been implicated in the development of gastric cancer andlymphoma. As mentioned above, H. pylori infection is one example wherecomplex triple therapies are required for eradication. One example isbased on Lansoprazole™ (30 mg b.d.) with amoxycillin (1000 mg b.d.) andmetronidazole (400 mg t.d.s.). It would be particularly useful thereforeto have available a simpler, less expensive therapy with gooderadication rates. In particular, it would be useful to produce ananti-H. pylori agent that negates the use of systemic antibiotics andreserves these for use against more serious (life-threatening) bacterialinfections.

H. pylori requires nickel ions for the assembly of active urease.However, nickel is a rare trace metal in the diet and is poorlyabsorbed. Therefore, its concentration in human blood and mucosa is verylow (2-11 nM). Due to the high requirement of H. pylori for this metalion, metal homeostasis is crucial for its survival in the gastricenvironment. The availability of the complete DNA sequence of the H.pylori genome has revealed the presence of multiple putative ion-bindingproteins and membrane transporters for divalent cations (Fulkerson etal, Conserved residues and motifs in the NixA protein of Helicobacterpylori are critical for the high affinity transport of nickel ions, TheJournal of Biological Chemistry, 273(1):235-241 1998). Nickel and othercations are imported by a relatively non-specific transport protein,namely NixA, which allows maximal accumulation of trace metals. However,these cations are enzyme inhibitors and urease is particularly sensitiveto certain cations (Perez-Perez et al., Effects of cations onHelicobacter pylori urease activity, release, and stability, Infectionand immunity, 62(1):299-302 1994). Therefore, the intracellularconcentration of cations is regulated in H. pylori by specific membraneproteins, namely CopA, CopA2 and CadA, that clear copper, cadmium andzinc from the cytoplasm, and to a lesser extent cobalt (Herrmann et al.,Helicobacter pylori CadA encodes an essential Cd(II)-Zn(II)-Co(II)resistance factor influencing urease activity. Molecular microbiology,33(3):524-5361999).

The use of dietary metals to treat gastrointestinal infections isdisclosed in WO 98/16248. The dietary metals are used in the form ofcomplexes with ligands such as citrate, maltol, lawsone and tropolone.

Perez-Perez et al, mentioned above, teaches only the decrease in ureaserelease caused by metal cations and, although specific therapies arepostulated, the document teaches that ions other than Ca²⁺ and Mg²⁺ arenon-specific.

EP-A-0770391 describes the use of a wide range of metals for thetreatment of a wide range of different medical conditions. There is nomention of selective treatment of H. pylori or of which metal ions wouldbe useful for this purpose.

WO 98/10773 describes the use of zinc or cobalt hyaluronate asantimicrobial agents. The polymeric, high molecular weight hyaluronicacid component is suggested as being essential for the pharmacologicalactivity and the data show that the compounds have no selectivity, beingactive against a wide range of different microorganisms.

Mobley et al, Helicobacter, Volume 4, Nov. 3, 1999, pages 162 to 169describe a mechanistic investigation into the effect of bismuth, Ni²⁺,Zn²⁺, Cu²⁺ and Co²⁺ on Hpn-negative mutants of H. pylori. Hpn was foundto have no effect on Co²⁺ tolerance in this academic study and noselectivity was attributed to any of the metals. The reference does notsuggest anything other than conventional bismuth salt treatments forinfections with H. pylori. The teaching of the document is away from Zn,Cu and Co, particularly so far as selectivity is concerned. Furthermore,there is nothing in Mobley et al that would suggest that cobalt hasactivity at about pH4 which is the pH in the environment for H. pyloriin vivo.

There exists a need for treatments of gastrointestinal infections whichare selective for the bacteria causing the infection and which do notadversely interfere with other bacteria present in the gut.

It has now been found that cobalt salts can be used to treatgastrointestinal infections with good selectivity. Surprisingly, cobaltshows excellent selectivity for H. pylori compared to othermicroorganisms.

Accordingly, the present invention provides the use of a cobalt salt inthe manufacture of a medicament for selectively treating and/orpreventing a gastrointestinal infection caused by H. pylori. Alsoprovided by the invention is a method of selectively treating and/orpreventing a gastrointestinal infection caused by H. pylori in a mammalwhich comprises the step of administering to a subject a therapeuticallyeffective amount of a cobalt salt.

Cobalt salts have been found to be particularly effective against H.pylori and especially selective for H. pylori ie, leaving many otherbeneficial bacteria present in the gut unaffected by the treatment.Thus, the invention is directed to the selective targeting of H. pyloriinfections.

The cobalt salt is preferably a cobalt (II) salt since it is well-knownthat cobalt (II) salts are readily available unlike cobalt (III) salts,and because there is concern over the long term safety of cobalt (III)salts.

The cobalt salt must be capable of providing cobalt ions (in hydratedform) at the site of the infection. It has been found that stronglycomplexing ligands such as EDTA can bind the cobalt ion so strongly thatactivity against the bacteria causing the infection is inhibited.Therefore, it is preferred that the cobalt salt comprises an anion whichdoes not complex too strongly with the cobalt ion. In addition, thiswill facilitate delivery of the cobalt ion to, and binding by, the mucuslayer. This is preferred as the gastric mucus layer is the environmentwhere H. pylori is resident. Preferably the anion is the anion of astrong acid (ie, having a pKa of less than about 4) and/or ismonovalent. However, anions of weaker acids such as the amino acids, forexample methionine and cysteine, may also be employed. Suitable anions,which must be substantially non-toxic and are preferably non-polymeric(ie, have a molecular weight of less than about 1000 daltons, preferablyless than 500 daltons, more preferably less than 200 daltons), includechloride, nitrate, sulphate, phosphate, carbonate, hydroxide, acetateand mixtures thereof (including salts containing two or more anions). Ofthese, chloride, carbonate, hydroxide and acetate are more preferred.Cobalt carbonate and hydroxide have the advantage of being substantiallyinsoluble in aqueous media at higher pH values but of forming solublesalts at lower pH values. Thus, these latter two salts can have theadvantage of gradually delivering the cobalt ions at the site ofinfection which is generally in an environment where the pH is low. Thiscould increase the residence time of the cobalt (II) ion in the gastricenvironment.

The cobalt salts of the invention preferably deliver cobalt ions inuncomplexed form (ie, such that Co(H₂O)₆ ²⁺ ions are formed when thesalt is in aqueous solution under gastric conditions, for example) or inthe form of complexes in which the cobalt ion is relatively weakly boundsuch that the complex at least partly dissociates to provide cobalt ionsin a form suitable for uptake into H. pylori at the site of infection.Thus, it is preferred that the anion in the cobalt salt and any ligandpresent in the salt or in the compositions of the invention does notbind to cobalt so strongly that cobalt ions (such as in hydrated form)are not available or are only available in relatively low amounts at thesite of the infection. In one embodiment, the invention is carried outin the absence of a bi- or poly-dentate complexing ligand for the cobaltions. Alternatively or additionally, the cobalt salt is preferably thesole di- or polyvalent metal species used in the invention. Preferably,the cobalt salt provides Co(H₂O)₆ ²⁺ ions as the major species (ie, thecobalt present as Co(H₂O)₆ ²⁺ ions is present in an amount of at least50% by weight based on the total amount of cobalt) when the salt isdissolved in excess deionised water (eg, greater then 10 molar excess,preferably greater than 100 molar excess) at 25° C. Alternatively, oradditionally, the cobalt ion/ligand complex preferably has an apparentaffinity constant K_(app) of less than 6, more preferably less than 5,wherein K_(app) is calculated at pH7 and 25° C. and is defined by theformula:

${\log\; K_{app}} = {{\log\; K_{i}} - {\log( {\frac{\lbrack H^{+} \rbrack}{K_{a1}} + \frac{\lbrack H^{+} \rbrack^{2}}{K_{a1}K_{a2}} + \ldots} )}}$where K_(i) is the stability of the species I formed between the ligandand metal, and K_(an) are the different acid dissociation constants ofthe ligand.

It will be appreciated that whether a ligand binds strongly to cobalt,in the context of the invention, will depend on thermodynamic andkinetic factors. Thus, anions forming complexes with cobalt with highaffinity may still be suitable for use in the invention if the cobaltion is kinetically labile. On the other hand, complexes of loweraffinity may be unsuitable for use in the invention if the kinetics ofthe dissociation are too slow. The ability of a given complex to providea sufficient amount of cobalt ions in vivo can be readily determined bythose skilled in the art on the basis of simple tests.

The present invention has the advantage of targeting the mucus layerwith the cobalt ion which may not occur with strongly bound cobaltcomplexes that would by-pass or not interact with the mucus layer. Themucus layer harbours H. pylori in the stomach. Also, the pH gradientwithin the mucus layer may facilitate the transfer of cobalt ionsthrough this layer to the bacteria. The cobalt ions may be used togetherwith one or more agents which facilitate targeting to the mucus layer.For example, the cobalt salt may be used together with agents such asthose selected from the group consisting of muco-adhesives andmucilagenous agents (e.g. acacia gum, tragacanth gum and methylcellulose), antimuscarinic agents (such as pirenzapine which binds tothe gastro-intestinal mucosa and delays gastric emptying), mucolyticagents (such as acetylcysteine, guaiphenesin or ammonium citrate),anti-gastritis agents and anti-ulcer agents which target the gastricmucus (such as carbenoxolone).

Currently, the most preferred anion is chloride and the preferred cobaltsalt is cobalt (II) chloride, optionally hydrated eg, CoCl₂.6H₂O.

The cobalt salts may comprise, or may be administered together with, aneutral or anionic ligand, although this is less preferred. The ligandsmay be useful in selectively delivering the cobalt ions to the site ofinfection. Suitable ligands include cysteine, methionine, and mixturesthereof.

The cobalt salts may also be used together with one or more ferric, zincor bismuth compounds or mixtures thereof. Suitable ferric compoundsinclude, for example, ferric maltol. Suitable zinc compounds includezinc citrate.

The cobalt salt, which may be a mixture of two or more different cobaltsalts, may be administered rectally or orally, preferably orally. Thesalt may be in the form of a liquid formulation, such as an aqueoussolution, a suspension or an emulsion, for example, or it may be in theform of a solid formulation, such as a capsule, pill or powder, forexample. The formulations containing the cobalt salt may containpharmaceutically acceptable excipients, diluents, carriers or otheradditives.

By “pharmaceutically acceptable” we include the normal meaning that thecarriers must be “acceptable” in the sense of being compatible with theactive ingredient (complex) and not deleterious to the recipientsthereof.

The composition may be in the form of a solid or liquid. Suitable solidcarriers include starch, lactose, dextrin and magnesium stearate. Liquidcarriers should be sterile and pyrogen free: examples are saline andwater.

Liquid formulations of the cobalt salts are particularly suitable fororal administration.

The cobalt salts may be formulated with a physiologically acceptablediluent or carrier for use as pharmaceuticals for veterinary or humanuse in a variety of ways. However, compositions in which the diluent orcarrier is other than a non-sterile solution in water are generallypreferred. Oral administration is, however, more generally to bepreferred for the treatment of H. pylori infections in humans and thecomplexes of the present invention may be given by such a route. Fororal administration in humans, it is more usual to use compositionsincorporating a solid carrier, for example starch, lactose, dextrin ormagnesium stearate. Such solid compositions may conveniently be shaped,for example in the form of tablets, capsules (including spansules), etc.However, liquid preparations are especially useful for oraladministration to patients who have difficulty in swallowing solidforms.

Preferably, the amount of cobalt salt which is required for thetreatment of gastrointestinal infections according to the inventionvaries from <1 to 300 mg, more preferably 1-100 mg, such as 1-50 mg ofcobalt per day. Single doses may be 1-50 mg, more preferably 1 to 30 mgper day, such as 20 mg per day, for example; doses in formulations whichare adapted for delayed or controlled release of cobalt ions may behigher than this, as will be appreciated by the skilled person. Asuitable dosage form comprises 10 mg of cobalt and such a dosage willtypically be administered twice daily for several weeks (eg 1 to 4weeks) in order to treat the infection. It will be appreciated by thoseskilled in the art that other dosage regimens may be equally applicablein the method of the invention.

Advantageously, the cobalt salts are formulated in a compositiontogether with an antibiotic and a pharmaceutically acceptable diluent orcarrier. Therefore, in a further embodiment, the present inventionprovides a pharmaceutical composition for use in the treatment ofgastrointestinal infections which comprises a cobalt salt and anantibiotic together with a pharmaceutically acceptable diluent orcarrier. Preferably, the composition also comprises a proton pumpinhibitor.

Another composition of the invention for use in the treatment and/orprevention of gastrointestinal infections comprises a cobalt salt and aproton pump inhibitor together with a pharmaceutically acceptablediluent or carrier.

The compositions of the invention may comprise a mixture of the cobaltsalt and the other pharmaceutically active substance or substances whichit contains (such as the antibiotic, for example). Alternatively, thecobalt salt and the other active substance or substances may be keptseparate from each other in the compositions and may even be packagedtogether but designed to be administered separately from each other.Therefore, the compositions of the invention may be in the form of a kitof parts.

The compositions of the invention may comprise more than one antibioticand/or more than one proton pump inhibitor.

Compositions for treating gastrointestinal infections containing a metalsalt, an antibiotic and a proton pump inhibitor, in so-called “tripletherapy” methods are known. Antibiotics and proton pump inhibitors,which are conventionally used in these compositions are suitable for usein the compositions of the present invention, provided that they areformulated in such a way as to be compatible with the cobalt salt.Suitable antibiotics include amoxycillin, metronidazole, clarithromycinand mixtures thereof, for example. Suitable proton pump inhibitorsinclude lansoprazole, omeprazole, pantoprazole and rabeprazole forexample. Proton pump inhibitors may be replaced by H₂ receptorantagonists, such as ranitidine and cimetidine, for example, in thepresent invention.

The compositions of the invention are preferably used for the selectivetreatment of gastrointestinal infection with H. pylori. Surprisingly,compositions containing cobalt salts are far more toxic for H. pylorithan other bacteria tested including those of the intestinal tract andboth Gram positive and Gram negative bacteria. This is an especiallyimportant finding given that one worrying side effect of presentantibacterial treatments for H. pylori is the eradication of commensalpro-biotic organisms of the gut leading to changes in normal intestinalflora with pathological implications such as antibiotic-induced colitis.Without wishing to be bound by theory, it is believed that theselectivity of cobalt for H. pylori may be due to the accumulation ofcobalt ions within H. pylori and the consequent action of cobalt as acumulative toxin. For this reason, it can be advantageous to providecobalt in the form of a controlled release formulation.

Cobalt salts also have the advantage of being selective for H. pylori atabout pH 4 (eg, pH 3 to 5), which is pH in the environment of H. pyloriin vivo.

Preferably, the cobalt salts have a selectivity for H. pylori such thatthe ratio of the MIC for cobalt against H. pylori to the MIC againstother bacteria commonly present in the intestinal tract, for exampleGram negative bacteria such as E. coli, is preferably greater than 10:1,more preferably greater than 100:1, most preferably greater than 200:1.MIC values are determined by the method described herein in the examplessection.

Therefore, the compositions of the invention and other medicaments foruse in the method of the invention may be adapted for delayed release ofthe cobalt ions. Suitable delayed release formulations (also termedcontrolled release formulations) are well-known to those skilled in theart. The delayed release of cobalt ions may be continuous and/or in theform of pulses. Preferably, the compositions release cobalt ions over aperiod of from 1 to 10 hours (e.g. from 3 to 10 hours, more preferably 4to 7 hours).

Controlled release of cobalt ions in the present invention may beachieved in a number of different ways. For example, the cobalt salt maybe formulated with a substance which delays gastric emptying. Suitablesubstances which delay gastric emptying include materials that bind tothe gastric mucosa (eg, aluminium compounds such as aluminium hydroxideor sucralfate, the hydrous basic aluminium salt of sucroseoctasulphate). Other substances that delay gastric emptying includeantispasmodics or anticholinergic agents such as propantheline andatropine. Substances which increase the viscosity of the formulation,eg, polysaccharide polymers such as alginate, may also be used to delaygastric emptying.

Alternatively, controlled release formulations may be derived byproviding a formulation that is capable of swelling on contact withwater. Thus, the formulation may comprise a cobalt salt together with apolymer capable of forming a polymeric hydrogel, preferably as a coatingaround the cobalt salt (and any other excipients). Such coatings can beformed, for example, from an anhydride copolymer, a cross-linking agent(eg, oxyethylene sorbitan monolaurate) and a plasticiser (eg, glyceryltriacetate). Styrene-maleic anhydride copolymers andpoly(methylvinylether/maleic anhydride) are suitable polymers. Thepolymer may be applied, for example, as a 1-20% (preferably 8-10%) byweight solution in a solvent mixture of ethylacetate and acetone(preferably about 65:35). Coating may be carried out, for example, byimmersion or spraying.

Other systems for controlled release of cobalt may also be used in theinvention. Suitable systems include coated beads, tablets with corescomprising microcrystalline cellulose, capsules comprising granuleshaving different release characteristics, multiple layer tablets, porousinert carriers, ion exchange resins and liquid gel preparations.

As described above, it is preferred that the cobalt in the cobalt saltsof the invention is in the cobalt (II) form. However, other forms ofcobalt may be employed, particularly if they may be converted to thecobalt (II) form at the site of the infection.

In the invention, it has been found that cobalt salts, such as cobaltouschloride, have antibacterial activity, at low concentrations, and arerelatively specific to the H. pylori genus and that cobaltous ions(Co²⁺) are the active species. Surprisingly, cobalt has been found to beactive even against the H. pylori strain deprived of its urease enzyme.

Cobalt specifically interferes with the oxidative mechanisms ofmammalian tissues and bacteria, which is an activity that is decreasedby the addition of cysteine (Orten and Bucciero, The effect of cysteine,histidine and methionine on the production of polycythemia by cobalt.Journal of biological chemistry, 176:961-968, 1948). Therefore, in thepresent invention, the cobalt salt may be used together with the weaklycomplexing amino acids cysteine and/or methionine. The presence ofcysteine and/or methionine at a weight ratio of 1:5 (cobalt:cysteineand/or methionine) has been found not to affect the MIC of cobaltagainst H. pylori and, therefore, the weight ratio of cysteine and/ormethionine to cobalt salt may range from 100:1 to 1:100 (such as 50:1 to1:50, more preferably 10:1 to 1:10).

Surprisingly, by measuring the MIC of cobaltous ion against H. pylori,it has been found that the metal was not only bacteriostatic (i.e.inhibitory of growth), but also bactericidal (i.e. rapidly decreasingviability).

The invention will now be described, by way of illustration only, withreference to the following non-limiting examples. In the examples andthroughout the specification all percentages are by weight unlessotherwise indicated.

EXAMPLES Methods

Solutions of metal salts were prepared by dilution of the metal salt,optionally together with the ligand, in ultra-high-purity water. The pHvalues were adjusted to 7 with concentrated sodium hydroxide. Thesolutions were γ irradiated for 20 to 30 minutes for sterilisation, andthen diluted ten fold in the bacterial growth medium to be used.

A gradient of concentration was obtained by two fold serial dilutions ofthe medium. The concentrations of metals were analysed by inductivelycoupled plasma spectrometry. Each dilution was inoculated with thedifferent bacteria to be tested, by multi-point inoculating on the agarmedia, or suspending in the broth media.

Media were incubated for Helicobacter pylori in a microaerobicatmosphere (gas jar containing a CampyGen® pack, 5% O₂-10% CO₂-85% N₂ byvolume) with constant shaking (orbital shaker, 140 rpm) at 37° C. forfive days.

The presence or absence of bacterial growth was reported within theconcentration gradient and the minimum concentration of complex in themedium that inhibits totally the bacterial growth (MIC) was determined.The growth on an agar plate was detected by the presence of colonies,and by turbidity in the broth media. The bacteria were identified by theGram morphology test. Helicobacter pylori growth was also confirmed bybiochemical test on the catalase, urease and oxidase activity.

Media and Bacteria Used:

Media Bacteria Gram − Bacteria Gram + BHI Helicobacter pyloriBrain-Heart infusion +2% horse serum Agar and Broth MH Helicobacterpylori Staphylococcus aureus Mueller-Hinton Echerichia coli Enterococcusfaecalis +2% horse serum Pseudomonas aeruginosa Enterococcus faeciumAgar and Broth Klebsiella Coagulase negative Staphylococcus ISOHelicobacter pylori Staphylococus aureus Isosensitest Echerichia coliEnterococcus faecalis +5% horse blood Pseudomonas aeruginosaEnterococcus faecium in Agar +2% horse serum Klebsiella Coagulasenegative in Broth Staphilococcus R&P Helicobacter pylori Reynolds andPenn defined medium for H. pylori Broth STH Helicobacter pylori StThomas' Hospital Fully defined medium for H. pylori Broth at pH4 or 7

FIG. 1 is a graph showing the inhibition of H. pylori growth by cobalt(cobaltous chloride) in the STH medium.

Example 1 Selectivity of Cobalt for Helicobacter pylori

Cobalt ion in the oxidation state +2 (Co²⁺) as cobalt chloride was foundto inhibit the growth of Helicobacter pylori, in vitro, with a minimuminhibitory concentration (MIC) of 0.06 mg/l.

The activity of Co²⁺ was specific for Helicobacter pylori (Table 1). Theactivity of Co²⁺ as cobalt chloride against other bacteria was higherthan the estimate limit between active and non-active metals, i.e. 32mg/l.

TABLE 1 MIC of cobalt chloride against other bacteria on Mueller-Hintonagar with 2% horse serum and isosensitest agar with 5% horse blood.Several strains of each genus were tested. Bacteria MIC (mg/l) Grampositive bacteria Staphylococcus aureus >32 Coagulase negativestaphylococcus Enterococcus faecalis Enterococcus faecium Gram negativebacteria Pseudomonas aeruginosa >32 Klebsiella Echerichia coli

Example 2 Activity of Cobalt Relative to Other Metal Ions

Other metal salts were tested for their action against H. pylori, andwere found to be poorly active (Table 2) with the exception of bismuth,which was active, but only at much higher levels than cobalt (II). Thissuggests a specific sensitivity of H. pylori to Co²⁺ compared to othermetals.

TABLE 2 MIC of metal ions against H. pylori on STH broth (NCTC 11637 andseven clinical isolates) metal ion tested MIC (mg/l) metal chloride:Fe³⁺ >32 Zn²⁺ >32 Mn²⁺ >32 Cu²⁺   16-32 Ni²⁺ >32 Co²⁺ 0.03-0.6 (median0.06) others: V⁴⁺ (vanadyl sulfate) >32 Cr²⁺ (chromium nitrate) >32 Mo⁶⁺(molybdenum oxide) >32 Bi³⁺ (bismuth nitrate)   4-8

Examples 3 to 12 Activity of Different Cobalt Salts

The MIC (mg/l) of different cobalt salts for H. pylori was determined attwo different pH values. The results are given in Table 3.

TABLE 3 MIC of different cobalt salts (Co (II) unless otherwiseindicated) Cobalt (Co) Salt pH 4-7* pH 7 Co chloride 0.03-0.4 0.06-0.4 Co nitrate 0.06 1 Co carbonate 0.03-0.125 0.125-1    Co bromide0.06-0.125    1-4 Co hydroxide 0.125    2-4 Co acetate 0.125 0.5-2   Cophosphate 4 0.8 Co sulfate 1 25 Co fluoride <5.4 9.5-1.9 Co (III)fluoride <4.5 15 *pH4-7 indicates that the pH of the media changesduring the experiment as the bacteria neutralise the starting acidicenvironment. This mimics better, therefore, the physiological situationwhere an ion may traverse the gastric mucus layer from an acidic pH to aneutral pH.

Example 13 Effect of Ligands on the Activity of Cobalt

The addition of the following ligands was found not to impair the MIC ofcobalt against H. pylori, although some more strongly complexing ligandsmay have reduced selectivity:

cysteine acetohydroxamic acid methionine alanine Hx maltol β-alanine Hxethylmaltol glutamine Hx lawsone phenylalanine Hx deferiprone (Hx =hydroxamate of the amino acid)

The strongly complexing ligands EDTA and folate inhibited the action ofcobalt against H. pylori and the strongly bound cobalt complex ofVitamin B₁₂ exhibited greatly reduced activity against H. pylori.

Example 14

Action of cobalt (cobaltous chloride) (Co) and amoxycillin (Ax) againstHelicobacter pylori 0 0.002 0.004 0.008 0.015 0.03 0.06 mg/l Ax0 + + + + + + − (control) 0.004 + + + + + + − 0.008 + + + + + + −0.0015 + + + + + + − 0.030 − − − − − − − 0.060 − − − − − − − 0.120 − − −− − − − mg/l Co (+) = growth in the medium (−) = inhibition of growthMIC of cobalt (no amoxycillin): 0.030 mg/l MIC of amoxycillin (nocobalt): 0.06 mg/l

The combination of cobalt with the antibiotic amoxycillin does notimpair the action of cobalt or amoxycillin against Helicobacter pylori.The MIC values are unchanged.

Example 15 Inhibition of Helicobacter pylori Growth by Cobalt (CobaltousChloride) in the STH Medium

FIG. 1 shows the results of this experiment as cfu/ml plotted againsttime (hours).

I₅₀=inhibition of 50% of the Helicobacter pylori inoculated by cobaltouschloride. Concentration of cobalt in the medium was 2.00 ppm.

Cobalt total inhibition is achieved in 8 hours and the cobalt I₅₀ levelis reached after 5 hours.

Example 16

The following is an example of a formulation for use according to theinvention:

CoCl₂. 6H₂O 100 mg Alginic acid 200 mg Sucrose 500 mg Al₂O₃  5 ml of 4%w/w water suspension in peppermint water

15 mg propantheline may be included in the formulation as anantispasmodic.

Example 17

The following is an example of a formulation for use according to theinvention:

CoCl₂·6H₂O 100 mg Microcrystalline cellulose 400 mg Hypromellose qvHypromellose phthalate Coating

Example 18

The following is an example of a formulation for use according to theinvention:

CoCO₃ 40 mg as cobalt Sulfacrate  1 g Minors qv

For dosing at 1-2 g every 4 hours.

Example 19

The data below indicate the selectivity of cobalt ions for H. pylori(Tables 4 and 5). In addition it should be noted that in Table 5 theactivity of cobalt against H. pylori is about 10 fold greater at pH 4than pH 7. Most anti-microbials are less active at acidic pH so such afinding is not necessarily to be expected.

TABLE 4 MIC and Selectivity Ratio of Metal Salts against PathogenicMicro-organisms Staphylococcus Average Pseudomonas Ent (aureus orSelectivity H. pylori Klebsiella E. coli aeruginosa faecalis OxfordStaph) Ratio Cu   24 >32 >32 >32 >32 >32 1.02   (16/32) 17.15^(a)1.75^(a) 17.15^(a) Zn >32/32 >32 >32 >32 >32 >32 0.76 17^(a) 1.7^(a)17^(a) Fe >32/>32 >32 >32 >32 >32 >32 1.00Mn >32/>32 >32 >32 >32 >32 >32 1.00 Ni >32/>32 >32 >32 >32 >32 >32 0.8922.89^(a) 22.89^(a) 22.89^(a) Co   0.06 >32 >32 >32 >32 >32 434(<0.03-0.125) 22.97^(a) 2.3^(a) 22.97^(a) Ag   0.13 — 0.11^(a) 0.11^(a)— 0.11^(a) 0.85 (<0.06-0.2) Footnotes to Table 4: ^(a)MIC resultsextrapolated from bacterial survival rates of organisms cultured inprotein broth and then transferred to phosphate-buffered saline. From:Zhao, Z H., Sakagami, Y., and Osaka, T. Relationship between residualmetal ions in a solution and the inhibitory capability of the metal ionsfor pathogenic bacterial growth. Bulletin of the Chemical Society ofJapan 71(4): 939-945; 1998. It should be noted that accuratecomparisonsbetween MIC results of different laboratories are difficultbecause conditions are not exactly replicated. However, these data ofZhao, et al are from experiments with liquid cultures rather than solid(agar) plates making conditions closer to those reported in the methoddescribed here-in. Indeed, the relative similarity between the twogroups for MIC data of most metals against most organisms supports theuse of this data for comparison. Average selectivity ratio = (Sum of MICvalues against all bacteria except H. pylori ÷ No. of MIC values) ÷ MICvalue for H. pylori.

TABLE 5 MIC of Cobalt salts against H. Pylori at pH 4 and pH 7 Strain 1Strain 1 Strain 2 Strain 2 Mean ratio Co compound (pH 4) (pH 7) (pH 4)(pH 7) pH 4:pH 7 Chloride 0.015 0.03/0.125 — —  0.5 & 0.12 Nitrate 0.050.69 0.055 >0.69   0.07 & 0.08 Carbonate 0.09 1.09 <0.02  1.09 0.048 &0.018 Bromide <0.04  1.12 0.09 4.49 0.036 & 0.02 Hydroxide 0.11 1.9 0.113.80 0.058 & 0.029 Acetate 0.11 2.03 0.11 1.01 0.054 & 0.11 Mean ratioof results at pH 4 against results at pH 7 is 0.095, suggesting that atpH 4 the efficacy of the cobalt ion is an order of magnitude moreefficacious than at pH 7

Example 20

In a tolerance study, six patients with H. pylori infection ingested alow dose of 20 mg/day cobalt, as cobalt chloride (10 mg b.d), for twoweeks. There were no reported adverse effects. In one patient the cobaltwas taken also with a proton pump inhibitor (Lansoprazole) while theremaining 5 took cobalt alone as capsules (4 patients) or a solution (1patient). 3 of these patients had gastritis and/or duodenitis with aduodenal ulcer also in one of these three. The other 3 had oesophagitisand/or normal stomach and duodenum.

1. A method of selectively treating a gastrointestinal infection causedby Helicobacter pylori (H. pylori) in a mammal, which comprises the stepof administering to a mammal a therapeutically effective amount of acobalt salt comprising an anion of an acid having a pKa of less thanabout 4 and/or which is monovalent, wherein the cobalt salt is selectivefor H. pylori at a pH of 3 to 5 and wherein the cobalt salt deliverscobalt ions in an uncomplexed form and wherein a therapeutic amount ofthe cobalt salt selectively targets H. pylori infections.
 2. The methodof claim 1, wherein the cobalt salt is a cobalt (II) salt.
 3. The methodof claim 2, wherein the cobalt salt is administered together with aproton pump inhibitor.
 4. The method of claim 1, wherein the cobalt saltis administered together with a proton pump inhibitor.
 5. The method ofclaim 1, wherein the cobalt salt is bactericidal for H. pylori.
 6. Themethod of claim 1, wherein the anion is selected from chloride, nitrate,sulphate, phosphate, carbonate, hydroxide, acetate and mixtures thereof.7. The method of claim 6, wherein the anion is chloride.
 8. The methodof claim 7, wherein the cobalt salt is hydrated cobalt chloride.
 9. Themethod of claim 1, wherein the composition further comprises one or moreagents selected from the group consisting of muco-adhesives,mucilaginous agents, antimuscarinic agents and mucolytic agents.
 10. Themethod of claim 1, wherein the cobalt salt is selective for H. pylorisuch that it is more toxic for H. pylori than other bacteria present inthe gut, wherein the bacteria are selected from the group consisting ofKlebsiella, coagulase negative staphylococcus, Enterococcus faecalis,Enterococcus faecium, and Escherichia coli.
 11. The method of claim 10,wherein the bacteria are Klebsiella, coagulase negative staphylococcus,Enterococcus faecalis, and Enterococcus faecium.
 12. The method of claim1, wherein the ratio of minimum inhibitory concentration for the cobaltsalt against H. pylori to the minimum inhibitory concentration of thecobalt salt against other bacteria normally present in the intestinaltract of mammals is greater than 10:1.
 13. The method of claim 1,wherein the amount of cobalt salt provides 1 to 100 mg of cobalt perday.
 14. The method of claim 1, wherein the cobalt salt is more toxic toH. pylori as compared to other bacteria in the gut.
 15. A method ofselectively treating a gastrointestinal infection caused by Helicobacterpylori (H. pylori) in a mammal, which comprises the step ofadministering to a mammal a therapeutically effective amount of apharmaceutical composition comprising a cobalt salt comprising an anionof an acid having a pKa of less than about 4 and/or which is monovalentand which is adapted for the delayed release of cobalt ions over aperiod within the range of 1 to 10 hours, wherein the cobalt salt isselective for H. pylori at a pH of 3 to 5 and wherein the cobalt saltdelivers cobalt ions in an uncomplexed form and wherein a therapeuticamount of the cobalt salt selectively targets H. pylori infections. 16.The method of claim 15, wherein the composition further comprises aproton pump inhibitor, or an H₂ receptor antagonist.
 17. The method ofclaim 15, wherein the cobalt salt is a cobalt (II) salt.
 18. The methodof claim 17, wherein the cobalt salt comprises an anion selected fromchloride, nitrate, sulphate, phosphate, carbonate, hydroxide, acetateand mixtures thereof.
 19. The method of claim 18, wherein the anion ischloride.
 20. The method of claim 15, wherein the cobalt salt isselective for H. pylori such that it is more toxic for H. pylori thanother bacteria present in the gut, wherein the bacteria are selectedfrom the group consisting of Klebsiella, coagulase negativestaphylococcus, Enterococcus faecalis, Enterococcus faecium, andEscherichia coli enterococcus, enterococcus faecium and Echerichia coli.21. The method of claim 20, wherein the bacteria are Klebsiella,coagulase negative staphylococcus, Enterococcus faecalis, andEnterococcus faecium.
 22. The method of claim 15, wherein the ratio ofminimum inhibitory concentration for the cobalt salt against H. pylorito the minimum inhibitory concentration of the cobalt salt against otherbacteria normally present in the intestinal tract of mammals is greaterthan 10:1.
 23. A method of selectively treating a gastrointestinalinfection caused by Helicobacter pylori (H. pylori) in a mammal, whichcomprises the step of administering to a mammal a therapeuticallyeffective amount of a pharmaceutical composition, wherein thecomposition comprises a cobalt salt and a proton pump inhibitor or an H₂receptor antagonist together with a pharmaceutically acceptable diluentor carrier and wherein said cobalt salt comprises an anion of an acidhaving a pKa of less than about 4 and/or which is monovalent, whereinthe cobalt salt is selective for H. pylori at a pH of 3 to 5 and whereinthe cobalt salt delivers cobalt ions in an uncomplexed form and whereina therapeutic amount of the cobalt salt selectively targets H. pyloriinfections.
 24. The method of claim 23, wherein the cobalt salt isselective for H. pylori such that it is more toxic for H. pylori thanother bacteria present in the gut, wherein the bacteria are selectedfrom the group consisting of Klebsiella, coagulase negativestaphylococcus, Enterococcus faecalis, Enterococcus faecium, andEscherichia coli.
 25. The method of claim 24, wherein the bacteria areKlebsiella, coagulase negative staphylococcus, Enterococcus faecalis,and Enterococcus faecium.
 26. The method of claim 23, wherein the ratioof minimum inhibitory concentration for the cobalt salt against H.pylori to the minimum inhibitory concentration of the cobalt saltagainst other bacteria normally present in the intestinal tract ofmammals is greater than 10:1.